Keratin bioceramic compositions

ABSTRACT

A malleable bone graft composition is described. The composition comprises: (a) keratose; (b) particulate filler; (c) antibiotic; and (f) water. The invention may be provided in sterile form in an container, and optionally lyophilized. Methods of treating a fracture with such compositions are also described.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/728,971, filed Oct. 21, 2005, the disclosure ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention concerns bioceramic bone graft compositions andmethods of using the same.

BACKGROUND OF THE INVENTION

Bone loss following bone fracture is a significant problem. Indeed, boneloss in combination with infection can lead to chronic osteomyelitis andnon-union. Since the first report of incorporating antibiotics into bonecement in 1970, many hospitals have adopted the practice forprophylactic treatment during arthroplasty, as well as for arrest ofchronic infections (McQueen M et al., Int Ortho 1987; 11:241-3; Fish D Net al., Am J Hosp Pharm 1992; 49: 2469-74; Hanssen A D and Osmon D R.Clin Ortho Rel Res 1999; 369(1): 124-38; Hanssen A D. J Arthroplas 2002;17(4S1): 98-101).

The use of antibiotic-impregnated poly(methylmethacrylate) (PMMA) beadshas also become widespread (Henry S L et al., Ortho Rev 1991; 20(3):242-7; Popham G J et al., Ortho Rev 1991; 20(4): 331-7; Klemm K W. ClinOrtho Rel Res 1993; 295: 63-76), although in the case of infectednon-unions or bony defects, this technology is typically used in twostage operations. In these cases, the defect or non-union site isdebrided as needed and the infection treated by placement ofantibiotic-impregnated PMMA beads into the defect site. In the secondstage, the beads are removed approximately six weeks later and a graftis used to repair the bone defect. The graft can be animal derived,allogenic, or autologous, such as COLLAGRAFT® bone graft matrix,demineralized bone matrix, or bone from the iliac crest, respectively.This two-stage methodology has also been widely adopted (Ueng S W N etal., J Trauma 1996; 40(3): 345-50; Ueng S W N et al., J Trauma1997;43(2):268-74; Chen C Y et al., J Trauma 1997; 43(5); 793-8;Swiontkowski M F et al., J Bone Joint Surg Br 1999; 81(B6):1 046-50).

In a logical progression of the technology, the two-stage method wassoon followed by a one-stage procedure. In this technique, antibioticsare combined with the bone graft material in order to limit theintervention to a single surgery. Ideally, the resident antibioticprovides local delivery for prophylactic treatment of infection whilethe graft provides the environment to grow new bone. This methodologyhas also been widely adopted and used both with human autologous andbovine grafts as well as synthetic grafts (Chan Y S et al., J Trauma1998; 45(4): 758-64; Chan Y S et al., J Trauma: Inj Inf Crit Care 2000;48(2): 246-55; Winkler H et al., J Antimicrobial Chemo 2000; 46: 423-8;Sasaki S and Ishii Y., J Ortho Sci 1999; 4: 361-9; McKee M D et al., JOrtho Trauma 2002; 16(9); 622-7).

These approaches are not without their limitations. For example, thetypical protocol for impregnation of antibiotic into PMMA is to heat thepolymer to form a melt, then to mix powdered antibiotic into the liquid.The antibiotic must be heat stable to withstand the PMMA melttemperatures, which is often not the case so the number of potentialantibiotics is limited. In addition, powdered antibiotic and liquid PMMAare not thermodynamically miscible; therefore the mixture is typicallynot homogeneous. This leads to uneven release of the antibiotic.Finally, the two-stage protocol subjects the patient to two surgeriesand thereby, increased risk.

Similar limitations exist when impregnating graft materials withantibiotic. Typical graft materials are donor bone, demineralized bonematrix, or synthetic ceramic substitutes (e.g. hydroxyapatite), amongothers. These biomaterials are often not compatible with the antibiotic,and the resulting composite is non-homogeneous. Whether one employsimpregnation into PMMA or a graft material, these methods, althoughclinically effective to some degree, are not controlled release systemsand are by no means optimized for therapeutic dosing of antibiotics.They are osteoconductive, and in the case of autologous bone arecertainly osteoinductive, but any approach that uses autologous bonesubjects the patient to another wound. This increases risk to thepatient and can lead to donor site morbidity, thereby compounding theoriginal problem (Silber, J S et al., Spine 2003; 28(2): 134-9).

Obviously, healing bone defects is a challenging area of orthopaedicmedicine. Current methods are not optimized for complete patientbenefit. Ideally, bony defects should be healed with a graft materialthat provides both an osteoconductive and osteoinductive environment,and controlled, effective antibiotic treatment in a biomaterial that canbe utilized in a single-stage operational protocol.

A recent review (Ludwig, S C et al., Eur Spine J 2000; 9(S1): S119-25)on the subject of bone graft substitutes listed the three most importantelements of the ideal product as:

-   -   1. Osteoconductive in that it provides a scaffold conducive to        vascular invasion, cell infiltration, and new bone formation;    -   2. Osteoinductive (i.e. capable of growth factor mediated        differentiation of precursor cells into osteoblasts); and    -   3. Capable of delivering cells that will form new bone matrix.        Any effective regeneration scheme must seek to optimize all        three of these parameters in order to recapitulate functional        bone. Accordingly, there is a continuing need for new        compositions useful as bone graft materials.

SUMMARY OF THE INVENTION

A first aspect of the invention is a malleable bone graft composition,comprising, consisting of or consisting essentially of:

(a) from 1 to 90 percent by weight keratose;

(b) from 1 to 90 percent by weight particulate filler (e.g. anosteoconductive filler);

(c) from 0.001 to 5 percent by weight antibiotic; and

(d) water to balance;

the composition having a viscosity of at least 3 centipoise at atemperature of 37° C.

in some embodiments the keratose is alpha keratose, gamma keratose, ormixtures thereof; in some embodiments the keratose is a mixture of alphakeratose and gamma keratose; in some embodiments the keratose comprisesfrom 10 to 90 percent by weight alpha keratose and from 90 to 10 percentby weight gamma keratose; in some embodiments the said keratose iscrosslinked keratose (e.g., produced by the process of combining thekeratose with transglutaminase in the presence of a calcium initiator).

In some embodiments the composition further comprises from 0.001 to 5percent by weight bone morphogenic protein.

In some embodiments the composition is sterile, and in some embodimentsthe composition is packaged in a sterile container.

A further aspect of the invention lyophilized or freeze-driedcomposition which upon reconstitution with water or saline solutionproduces a composition as described herein.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below. The disclosures of all United States patent referencescited herein are to be incorporated by reference herein in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Keratin biomatenral scaffold (a) was formed spontaneously by aself-assembly mechanism. Notice the fibrous architecture, high porosity,and homogeneity similar to that of native ECM (b; bladder submucosa ECMshown). This is in dramatic contrast to synthetic scaffolds that claimto have “interconnected” pores (c). These types of synthetic scaffoldsare difficult to seed at best and must rely on degradation of the matrixfor tissue infiltration. This results in slower, less complete healing.

FIG. 2. Viscosity curves for α-keratose (bottom curve) and α-SCMK (topcurve). Samples were formulated at 5 weight percent and below in RLsolution to provide high porosity. Low solids content hydrogels aredesired to provide biocompatibility and to accommodate the in growth ofosteoblasts.

FIG. 3. SEM micrographs of keratose formulations (as defined in Table1). Samples were created from lyophilized solutions in order toinvestigate the underlying microstructure of the hydrogels. Those gelsshowing the most fibrous microstructure also demonstrated the greatestincrease in apparent viscosity.

FIG. 4. Kill curves for an antibiotic containing keratin biomaterial.These data demonstrate the efficacy of a keratin biomaterial DDS on S.aureus. Effective arrest of the bacteria was noted at each concentrationof Cefazolin.

FIG. 5. Release kinetics for KBAP formulations containing Cefazolin weremeasured in a modified Franz diffusion cell (a). The antibiotic wassimply added to the keratin hydrogel and was not encapsulated orchemically conjugated. Consequently, the release kinetics show rapiddelivery in the first 24 hours, followed by much lower release duringthe subsequent 3 days. Encapsulation and conjugation methods arecurrently being developed to provide an MIC for up to 2 weeks.

FIG. 6. Growth of bovine osteoblasts in the presence of six differentKBAP formulations compared to control conditions (media alone). Opticaldensity values (Y axis) are proportional to the total number of viablecells. These data suggest that KBAP formulation nos. 3, 4, 5, 8, and 9are compatible with osteoblasts (p>0.05, n=5). Formulation nos. 6 and 7reached near significance with p values of 0.030 and 0.041, respectively(n=5).

FIG. 7. Micrgraphs showing the effect of the KBAP extract on osteoblastmigration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The compositions described herein are intended for use in the treatmentof human subjects (including males and females, and including infant,juvenile, adolescent, adult and geriatric subjects) as well as animalsubjects, particularly other mammalian subjects such as dogs, cats,horses, etc., for veterinary purposes.

“Bone” as used herein includes any bone, such as: the pelvis; long bonessuch as the tibia, fibia, femur, humerus, radius, and ulna, ribs,sternum, clavicle, etc.

“Fracture” or “break” as used herein with respect to bones includes anytype thereof, including open or closed, simple or compound, commirnutedfractures, and fractures of any location including diaphyseal andmetaphyseal. “Fracture” as used herein is also intended to includedefects such as holes, gaps, spaces or openings, whether naturallyoccurring or surgically induced (e.g., by surgical removal of undesiredtissue from bone).

“Antibiotic” as used herein includes any suitable antibiotic, includingbut not limited to cefazolin, vancomycin, gentamycin, erythromycin,bacitracin, neomycin, penicillin, polymycin B, tetracycline, biomycin,chloronmycetin, streptomycin, ampicillin, azactam, tobramycin,clindamycin, gentamicin and combinations thereof. See, e.g., U.S. Pat.No. 6,696,073. In some embodiments the antibiotic is preferably a watersoluble antibiotic.

“Particulate fillers” used to carry out the present invention can beformed from any suitable biocompatible material, such as a ceramic. Insome embodiments, the particulate filler is preferably osteoconductive.Examples of suitable materials from which the filler may be formedinclude but are not limited to tetracalcium phosphate, tricalciumphosphate, calcium alkali phosphate ceramic, calcium phosphorus apatite,bioglass, calcium carbonate, calcium hydroxide, calcium oxide, calciumfluoride, calcium sulfate, magnesium hydroxide, hydroxyapatite, calciumphosphorus apatite, magnesium oxide, magnesium carbonate, magnesiumfluoride, collagen, allograft bone, other resorbable biocompatiblematerials and mixtures thereof See, e.g., U.S. Pat. No. 6,869,445;5,281,265. In some embodiments the particulate filler compriseshydroxyapatite, tricalcium phosphate, or a mixture thereof.

The particulate filler content of the composition of the presentinvention may be in a range from about 0.1 percent to about 200 percentof the keratin content of the composition. In some embodiments, theparticulate filler content of the composition may be in a range fromabout 10 percent to about 100 percent of the keratin content. In otherembodiments of the invention, the particulate filler content of thecomposition may be in a range from about 20 percent to about 90 percentof the keratin content. In further embodiments, the particulate fillercontent may be in a range from about 40 percent to 80 percent of thekeratin content. In additional embodiments, the particulate fillercontent of the composition may be in a range from about 25 percent toabout 50 percent of the keratin content. As an example, in oneembodiment, when the keratin concentration in 100 g of gel is 20 percent(i.e., 20 g keratin per 80 g water) then the particulate filler contentmay be in a range from about 2 g to about 20 g.

In particular embodiments, the composition of the present invention hasa consistency similar to toothpaste or modeling clay. Further, inrepresentative embodiments, the viscosity of the composition is fluidand malleable and able to hold a form or shape without a supportingstructure.

The composition of the present invention may be provided to the user ina dry form, which can be rehydrated for later use.

Keratin materials. Keratin materials are derived from any suitablesource including but not limited to wool and human hair. In oneembodiment keratin is derived from end-cut human hair, obtained frombarbershops and salons. The material is washed in hot water and milddetergent, dried, and extracted with a nonpolar organic solvent(typically hexane or ether) to remove residual oil prior to use.

Scheme 1 below provides general representations of (a) oxidation and (b)reduction of disulfide crosslinks in keratin. These reactions cleave thesulfur-sulfir bond in cystine residues, thereby destroying thesuperstructure and rendering the keratins soluble in the reaction media.

Keratose Fractions. Keratose fractions are obtained by any suitabletechnique. In one embodiment they are obtained using the method ofAlexander and coworkers (P. Alexander et al., Biochem. J. 46, 27-32(1950)). Basically, the hair is reacted with an aqueous solution ofperacetic acid at concentrations of less than ten percent at roomtemperature for 24 hours. The solution is filtered and thealpha-keratose fraction precipitated by addition of mineral acid to a pHof ca. 4. The alpha-keratose is separated by filtration, washed withadditional acid, followed by dehydration with alcohol, and then driedunder vacuum. Increased purity can be achieved by redissolving thekeratose in a denaturing solution such as 7M urea, aqueous ammoniumhydroxide solution, or 20 mM tris base buffer solution,re-precipitating, re-dissolving, dialyzing against deionized water, andre-precipitating at pH 4.

The gamma-keratose fraction remains in solution at pH 4 and is isolatedby addition to a water-miscible organic solvent such as alcohol,followed by filtration, dehydrated with additional alcohol, and driedunder vacuum. Increased purity can be achieved by redissolving thekeratose in a denaturing solution such as 7M urea, aqueous ammoniumhydroxide solution, or 20 mM tris buffer solution, reducing the pH to 4by addition of a mineral acid, removing any solids that form,neutralizing the supernatant, re-precipitating the protein with alcohol,re-dissolving, dialyzing against deionized water, and reprecipitating byaddition to alcohol. The amount of alcohol consumed in these steps canbe minimized by first concentrating the keratose solution bydistillation.

In use the compositions may be rehydrated if necessary, and used totreat fractures in a subject (e.g. filling bone defects) in accordancewith known techniques by contacting the composition to the fracture in atreatment-effective amount. Fractures may be of any bone, including butnot limited to: ethmoid, frontal, nasal, occipital, parietal, temporal,mandible, maxilla, zygomatic, cervical vertebra, thoracic vertebra,lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus,radius, ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium,pubis, femur, tibia, fibula, patella, calcaneus, tarsal bones ormetatarsal bones, etc. Indeed the compositions may be used for anysuitable purpose for which bone graft or osteogenic implants are used,as described in U.S. Pat. No. 6,863,694 to Boyce et al.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXPERIMENTAL

In this study, a keratin bioceramic antibiotic putty (KBAP) thatprovides osteoconductivity, osteoinductivity, and controlled antibioticrelease is tested. The putty is comprised of a keratin hydrogel withceramic filler and antibiotic. The KBAP is malleable and can be formedinto shapes and pressed into a bony defect site with no additionalpreparation. It provides immediate and prophylactic antibiotic releaseas well as an osteoconductive and osteoinductive environment for boneregeneration in a single-stage operational protocol.

Keratin hydrogel is a proteinaceous network that is highly hydrated;therefore any water soluble antibiotics (e.g. Cefazolin, Gentamicin, andVancomycin) can be used. The hydrophilicity of the hydrated keratinpromotes cell attachment and in growth. The ceramic component may havethe osteoconductive properties of products currently on the market(e.g., COLLAGRAFT®), but may not require aspirated bone marrow. Thekeratin matrix provides a highly biocompatible environment, as keratinsare a class of proteins that elicit one of the lowest foreign bodyreactions among all biomaterials (Ito H et al., Kobunshi Ronbunshu1982;39(4):249-56; Blanchard C R et al., U.S. Pat. No 6,461,628. Oct. 8,2002; Tachibana A et al., J Biotech 2002;93:165-70).

When processed correctly, keratin proteins have a unique capability ofmolecular self-assembly, a process by which they reconstruct somesemblance of their original tertiary structure (Sauk J J et al., J CellBio 1984;99:1590-7; Thomas H et al., Int J Biol Macromol 1986;8:258-64;van de Löcht M. Melliand Textilberichte 1987;10:780-6). This is aparticularly useful characteristic for a bone graft substitute for tworeasons. First, self-assembly results in a highly regular structure withreproducible architectures, dimensionality, and porosity. Second, thefact that these architectures form of their own accord under benignconditions allows for the incorporation of cells as the matrix isformed. These two features are critically important to any system thatattempts to mimic the native ECM. The keratin scaffold shown in FIG. 1was prepared by spontaneous self-assembly of a hydrogel and demonstratesthe type of architecture conducive to cell infiltration and tissueregeneration.

Native ECM is a regular structure created around the cells, by thecells. In a tissue damage scenario, the ECM is an interactive medium forcell recruitment, growth, and differentiation, leading to the formationand maturation of new functional tissue. The ECM helps to orchestratethese processes by providing architectural support, growth factordelivery, and sites of molecular recognition whereby cells can bind andreceive information.

Cellular recognition is facilitated by the binding of cell surfaceintegrins to specific amino acid motifs of the ECM (Buck C A and HorwitzA F. Annu Rev Cell Biol 1987;3:179-205; Akiyama S K. Hum Cell 1996;9(3):181-6). The predominant ECM proteins are collagen and fibronectin, bothof which have been extensively studied with regard to cell binding(McDonald J A and Mecham R P (editors). Receptors for extracellularmatrix (1991). Academic Press, San Diego). Fibronectin contains severalregions that support attachment by a wide variety of cell types. Mouldet al showed that in addition to the widely knowArginine-Glycine-Aspartic Acid (RGD) motif, the “X”-Aspartic Acid-“Y”motif on fibronectin is also recognized by the integrin α4β1 where Xequals Glycine, Leucine, or Glutamic Acid and Y equals Serine or Valine(Mould A P et al., J Biol Chem 1991;266(6):3579-85). Inexpensive,biocompatible scaffolds from keratin biomaterials contain these samebinding motifs.

A recent search of the NCBI protein database revealed sequences for 71discrete, unique human hair keratin proteins (Data from the NationalCenter for Biotechnology Information (NCBI) database.http://www.ncbi.nlm.nih.gov). Of these, 55 are from the high molecularweight, low sulfur, alpha-helical family. This group of proteins isoften referred to as the alpha-keratins and is responsible for impartingtoughness to human hair fibers. These alpha-keratins have molecularweights greater than 40 kDa and an average cysteine (the main amino acidresponsible for inter- and intramolecular protein bonding) content of4.8 mole percent. Importantly, analysis of the amino acid sequences ofthese alphakeratin proteins showed that 78% contain at least onefibronectin-like integrin receptor binding motif, and 25% contain atlease two or more. A recent paper has highlighted the fact that thesebinding sites are present on keratin biomaterials by demonstratingexcellent cell adhesion to a keratin foam (Tachibana A et al., J Biotech2002;93:165-70). Although this paper uses fibroblasts to demonstrate theprinciple, the osteoconductivity of a keratin bioceramic was laterdemonstrated by these same authors (Tachibana A et al., Biomaterials2005;26(3):297-302).

Some studies are beginning to show mounting evidence that a number ofgrowth factors are present in end-cut human hair, and that the keratinsmay be acting as a highly effective delivery matrix. It has been knownfor more than a decade that growth factors such as bone morphogeneticprotein-4 (BMP-4) and other members of the transforming growth factor-β(TGF-β) superfamily are present in developing hair follicles (Jones C Met al., Development 1991; 111: 531-42; Lyons K M et al., Development1990; 109: 833-44; Blessings M et al., Genes and Develop 1993; 7:204-15).39-41 In fact, more than 30 growth factors and cytokines areinvolved in the growth of a cycling hair follicle (Stenn K S et al., JDermato Sci 1994; 7S: S109-24). Many of these molecules have a pivotalrole in the regeneration of a variety of tissues (Clark R A F (editor).The molecular and cellular biology of wound repair (1996) Plenum Press,New York). It is highly probable that a number of growth factors becomeentrained within human hair when cytokines bind to stem cells residingin the bulge region of the hair follicle (Panteleyev A A et al., J CellSci 2001; 114: 3419-31). We have recently analyzed extracts of humanhair and shown the presence of growth factors such as vascularendothelial growth factor (VEGF) in these samples. We are currentlyassaying keratin biomaterials for the presence of several other growthfactors including BMP, TGF-β, and nerve growth factor.

The preceding discussion demonstrates several key advantages that theKBAP has over conventional bone graft substitutes, and the ways in whichwe are leveraging our expertise to achieve the objective of developing asuperior product. A keratin biomaterial with antibiotic filler providessustained antibiotic release and has the potential to achieve this goalfor the following reasons:

-   -   Keratin biomaterials are easily obtained and processed    -   Keratins are highly biocompatible    -   Keratins self-assemble into architectures that are conducive to        cell attachment and growth    -   Keratins contain sites of cellular recognition and are effective        ECM surrogates    -   Keratins are able to act as encapsulants or conjugates of drug        compounds such as antibiotics and control their release kinetics    -   Keratin biomaterials contain growth factors such as BMP that        modulate cell growth and differentiation and therefore have the        potential to impart osteoinductivity        Results        1. Prepare a Bone Graft Putty Using a Keratin Matrix with a        Mineral Component that can Incorporate Antibiotic Microcapsules.

There are many published methods that describe the extraction ofkeratins from hair fibers. In general, there are three procedures usedto chemically break down the resilient structure of the hair fiber andimpart aqueous solubility to the cortical keratin proteins ofinterest: 1) oxidation, 2) reduction, and 3) sulfitolysis (seek e.g.,Crewther W G et al., Advances in protein chemistry (1965). Anfinsen C BJr., Anson M L, Edsall J T, and Richards F M (editors). Academic Press.New York:191-346; Goddard D R and Michaelis L., J Bio Chem 1934; 106:605-14; Kelley R J et al., PCT Patent Application No. WO 03/011894;Zackroff R V and Goldman R D. Proc Natl Acad Sci 1979; 76(12): 6226-30).Efficient extraction depends first on breaking the disulfide bonds usingone of these three methods, and second on gently denaturing the freeproteins and affecting their dissolution. The importance of this secondstep cannot be over emphasized due to the existence of a competingreaction, hydrolysis of the peptide bonds in the keratin backbone.Hydrolysis of the protein backbone destroys many of the usefulproperties of keratins and must be avoided. We have evaluated all threeof these methods as described more fully below, with the goal ofcreating a malleable putty that can be used to repair bony defects.

Oxidation: Human hair was obtained from a local salon, washed with milddetergent (Fisher Scientific, Pittsburgh, Pa.), degreased with ethylether (Sigma-Aldrich, St. Louis, Mo.), and dried in air. In a typicalreaction, 20 grams of clean, dry hair was treated with 400 mL of a 2weight/volume (w/v) % solution of peracetic acid (PAA, Sigma-Aldrich,St. Louis, Mo.) in deionized (DI) water. The oxidation was conducted ina closed polypropylene container maintained at 37° C. for 12 hours withgentle agitation. The oxidized hair was recovered and rinsed withcopious amounts of DI water. The wet, oxidized hair was extracted withsuccessive volumes of 0.2M tris base (Sigma-Aldrich, St. Louis, Mo.),0.1M tris base, and DI water (500, 500, and 1000 mL, respectively). Theextracts were combined and the α-keratose precipitated by drop wiseaddition of 12M hydrochloric acid (HCL; Fisher Scientific, Pittsburgh,Pa.) to a final pH of 4.2. The α-keratose was re-dissolved in 20 mM trisbase with 20 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich,St. Louis, Mo.), re-precipitated by drop wise addition of HCL to a finalpH of 4.2, and again re-dissolved in tris base +EDTA. The resultingprotein solution was dialyzed against DI water for three days with twicedaily water changes (LMWCO 12.4K; Sigma-Aldrich, St. Louis, Mo.). Afterdialysis, the o-keratose powder was isolated by reducing the liquidvolume via vacuum distillation at 50° C. and freeze-drying theconcentrate. This sample was formulated at 5, 4, 3, 2, 1, and 0.5 weightpercent in Ringer's lactate (RL) and analyzed for viscosity on aBrookfield cone and plate viscometer (Brookfield Engineering,Middleboro, Mass.) at 37° C. The viscosity data are shown in FIG. 1.

Reduction: A second sample of keratin was obtained using a differentextraction protocol. Human hair was obtained from a local salon, washedwith mild detergent, degreased with ethyl ether, and dried in air. In atypical reaction, 20 grams of clean, dry hair was treated with 400 mL ofa 1.0M thioglycolic acid (Sigma-Aldrich) in DI water that had beentitrated to pH 10.2 using saturated sodium hydroxide solution (FisherScientific). The reduction was conducted in a closed polypropylenecontainer maintained at 37° C. for 12 hours with gentle agitation. Thereduced hair was recovered and rinsed with copious amounts of DI water.The wet, reduced hair was extracted with three successive 500 mL volumesof 0.1M tris base with 0.1M thioglycolic acid. The extracts werecombined and the α-kerateine precipitated by drop wise addition of 12MHCL to a final pH of 4.2. The α-kerateine was re-dissolved in 20 mM trisbase with 20 mM EDTA, re-precipitated by drop wise addition of HCL to afinal pH of 4.2, and again re-dissolved in tris base +EDTA. The proteinsolution was dialyzed against DI water for three days with twice dailywater changes (LMWCO 12.4K). After dialysis, the α-kerateine wasderivatized by reacting the cysteine residues with iodoacetic acid(Sigma-Aldrich) by adding 0.25 mg per mL of dialyzate. The reaction wasperformed in a closed polypropylene container maintained at 37° C., pH9.0, with occasional stirring over 24 hours. Excess lodoacetic acid andother contaminants were removed by dialysis against DI water (LMWCO12.4K). The dialyzate was concentrated by reduced pressure evaporationand the α-s-carboxymethylkerateine (α-SCMK) obtained by freeze-drying.Solutions of α-SCMK at 5, 4, 3, 2, 1, and 0.5 weight percent in RL wereprepared and analyzed for viscosity as described previously. These dataare also shown in FIG. 2.

Although these data trend toward acceptable viscosity values,formulating the hydrogels at 10 weight percent or less is desired tomaintain biocompatibility and provide a porous matrix that can bepopulated by osteoblasts. Biomaterials with low porosity or pores thatare too small (i.e. <85% and <100 μm, respectively) are difficult forcells to populate without first degrading them; this slows healing andcan lead to fibrosis. The viscosities shown in FIG. 1 for formulationsbelow 5% keratin were deemed to be unacceptably low. It was determinedthat the extraction conditions employed resulted in excessivehydrolysis, consequently, we modified our oxidation protocol to minimizethis side reaction.

Oxidation (low hydrolysis method): In a typical procedure using thisprotocol, 50 grams of clean, dry hair was treated with 1,000 mL of a 2weight/volume (w/v) % solution of PAA in DI water. The oxidation wasconducted in a closed polypropylene container maintained at 37° C. for12 hours with gentle agitation. The oxidized hair was recovered andrinsed with copious amounts of DI water. The wet, oxidized hair wasextracted with 1,000 mL of 0.1M tris base and subsequently extractedwith successive 1,000 mL volumes of DI water. The extracts were combinedand concentrated 10-fold by reduced pressure evaporation at 50° C. Theα-keratose was precipitated by drop wise addition of the concentratedsolution to cold ethanol. The precipitate was re-dissolved in a minimumamount of DI water and re-precipitated by drop wise addition of 12M HClto a final pH of 4.2. The α-keratose was isolated by centrifugation,re-dissolved in DI water, adjusted to a pH of 7.0, dialyzed against DIwater for three days with twice daily water changes (LMWCO 12.4K),concentrated, and freeze dried. Solutions of the low hydrolysis (LH)α-keratose were prepared at 10 and 5 weight percent in phosphatebuffered saline (PBS) and analyzed for viscosity as describedpreviously. At 10 weight percent, the viscosity was too high to bemeasured by the viscometer. At 5 weight percent, the viscosity (analyzedat lower torque than previous measurements) was 460 centipoise at 37° C.From these data, we concluded that the LH method substantially reducedhydrolysis during keratin extraction and downstream processing.

Recognizing that additional improvements could potentially be made inour keratin production process, a small change was made to the LHextraction protocol described above. Rather than separate α- andγ-keratose by isoelectric precipitation, the crude extract was dialyzedto remove trace contaminants and residual processing chemicals, and thedialyzate concentrated and freeze dried. This results in a mixture ofboth α- and γ-keratose that has demonstrated improved viscoelasticproperties. In addition, we attempted to improve the viscosity of thehydrogel formulation by crosslinking the keratose mixture. The detailsof this method are as follows: In a typical procedure, 50 grams ofclean, dry hair was treated with 1,000 mL of a 2 weight/volume (w/v) %solution of PAA in DI water The oxidation was conducted in a closedpolypropylene container maintained at 37° C. for 12 hours with gentleagitation. The oxidized hair was recovered and rinsed with copiousamounts of DI water. The wet, oxidized hair was extracted with 1,000 mLof 0.1M tris base and subsequently extracted with successive 1,000 mLvolumes of DI water. The extracts were combined and concentrated 10-foldby reduced pressure evaporation at 50° C. The concentrated solution wasdialyzed against DI water (LMWCO 12.4K), concentrated, and freeze dried,5 w/v % solutions of α+γ-keratose and α-keratose in DI water wereprepared and their viscosities measured. These solutions were alsoreacted with a solution of transglutaminase (1 mg/mL; Sigma-Aldrich, St.Louis, Mo.) in an attempt to further increase their viscosity throughlutamine-lysine crosslinking (both amino acids are prevalent inkeratins). Interestingly, while the initial viscosity of theα+γ-keratose samples was lower than the α-keratose, the former achievedhigher viscosity than the latter after only one hour of incubation at37° C. Based on this observation, several formulations were prepared andcrosslinked with transglutaminase, and the viscosities of the resultinggels measured. These data are shown in Table 1.

These data must be interpreted carefully as the formulations containedless keratose than would normally be used for a KBAP formulation simplyto ensure their viscosities were within the range of our equipment.Also, addition of the transglutaminase and a small amount of calciuminitiator decreased the apparent weight percent keratose. These datasuggest that in forming a high viscosity hydrogel a mixture ofα+γ-keratose and the use of transglutaminase crosslinking may be useful.Qualitatively, the α-keratose solution decreased in viscosity after 1hour of incubation at 37° C. while a similar α+γ-keratose solutionincreased. This phenomenon may be due to complex formation between thealpha and gamma forms of keratin. TABLE 1 Viscosity of keratoseformulations measured at 30 rpm and 37° C. Transglutaminase Vol. ofkeratose (vol. of 1 mg/mL Viscosity (cP) No. Type solution usedsolution) Initial After 1 hr @ 37° C. 1 1% α-keratose 400 μL 100 μL 2.801.36 2 1% α-keratose 300 μL 300 μL 2.80 1.23 3 1% α + γ-keratose 400 μL200 μL 1.71 2.24 4 1% α + γ-keratose 300 μL 200 μL 1.71 2.69 5 2% α +γ-keratose 300 μL 0 μL 2.43 1.86 6 2% α + γ-keratose 300 μL 200 μL 2.431.93 7 5% α + γ-keratose 800 μL 100 μL 5.72 3.72 8 5% α + γ-keratose 800μL 200 μL 5.72 3.08 9 5% α + γ-keratose 700 μL 200 μL 5.72 2.65 10 5%α + γ-keratose 700 μL 400 μL 5.72 3.03

In addition to viscosity analysis, the underlying microstructure of thefirst six of these gel formulations was investigated. Each formulationwas recovered from the viscometer and freeze dried. The structures ofthe resulting samples were characterized by scanning electron microscopy(SEM; Model S-2600N; Hitachi High Technologies America, Inc.,Pleasanton, Calif.) and are shown in FIG. 3. These images show thefibrous nature of the hydrogels and demonstrate the effect of thismicrostructure on viscosity. Formulation no. 4, for example,demonstrated one of the largest increases in viscosity after enzymecrosslinking; it also shows the most developed fibrous architecture. Webelieve the fibrous architecture is mediated by a process of molecularself-assembly, a unique characteristic of keratin-based biomaterials.

2. Optimize and Validate the Antibiotic Formulation

In order to demonstrate antibiotic delivery, we conducted an initialinvestigation using the keratin as a drug carrier. In these experiments,Cefazolin sodium was dissolved in aqueous a-kerateine solution at targetconcentrations of 0, 10, 20, 40, and 80 μg. The solutions werelyophilized and the solid samples ground into fine powders. The powderswere formed into 500 mg disks using a manual press. The disks were usedto generate kill curves for suspensions of Staplylococcits aureus ATCC29213. Mueller Hinton broth cultures were prepared from an overnightsheep blood agar (SBA) culture of S. aureus so as to containapproximately 105 cfu per mL final concentration. Keratin disks wereplaced into culture tubes containing 2 mL final volume of S. aureusculture and incubated at 37° C. A 10 μL aliquot of each culture wasremoved at 0, 4, and 8 hours, diluted into 10 mL of saline, of which 100μL were plated onto SBA using a glass spreader and inoculatingturntable. Each concentration of antibiotic was tested in triplicate.The average number of colonies was calculated from the triplicatesamples and compared to a positive control culture without antibioticand a negative control culture without organisms. These data, shown inFIG. 4, demonstrate the efficacy of the keratin biomaterial DDS.

In a more recent experiment, a high viscosity KBAP formulation (LHmethod) was used to incorporate Cefazolin at 1000, 500, and 250 iigantibiotic by vortexing. The gel was cross-linked using 100 μl of 0.1%transglutaminase at 37° C. for 1 hour and the formulation waslyophilized to form a free standing disc. The Cefazolin release from theKBAP disk was measured in PBS using a modified Franz diffusion cell).The pellet (15 mg) was placed in the donor compartment, while theacceptor compartment was filled with PBS. The donor compartment wasseparated from the acceptor compartment by a cellulose membrane (100 gimpore size). This diffusion cell was then placed in an incubator at 37°C. The solution in the acceptor compartment was periodically removed andreplaced with and equal amount of fresh buffer solution. The Cefazolinreleased through the cellulose membrane was analyzed by UVIisspectrometry (Thermo Spectronic, USA, UV/VIS) at 285 nm.

As shown in FIG. 5, the release curves revealed a controlled release ofCefazolin from the KBAP formulation. Approximately 60˜70% of theincorporated antibiotic was released from KBAP in one day and sustainedthe release for 4 days.

3. Biocompatibility, Osteoconductivity, and Osteoinductivity in Vitro.

A sample of bovine osteoblasts was reconstituted from frozen stock byrapid thawing and plating onto a 10 cm tissue culture dish. The cellswere cultured in 10 mL of low glucose DMEM containing 10% FBS(Invitrogen, Carlsbad, Calif.) with 0.05 mg/mL ascorbic acid(Sigma-Aldrich, St. Louis, Mo.) and antibiotics added. Media was changedthree times per week and the cells were expanded by subculturing every3-5 days. After at least three subculture cycles (“passages”), the cellswere trypsinized and seeded in 96-well tissue culture plates at adensity of approximately 3,000 cells per well. To each of five replicatewells was added approximately 1 mg of one of the KBAP formulations shownin Table 2. The KBAP had been lyophilized and gamma sterilized prior toplacing in the media. After approximately 72 hours of incubation at 37°C., 5% CO2, and 95% humidity, 200 μL of3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT; 1mg/mL; Signa-Aldrich, St. Louis, Mo.) was added to the culture wells,the cells were incubated for 4 hours, and the metabolic reductionproduct, MTT formazan, dissolved in a known amount of dimethylsulfoxide.The number of cells was measured indirectly by measuring the colorintensity of metabolically reduced MTT using a microplate reader at 540nm (model E1x800; Bio-Tek Instruments, Inc.; Winooski, Vt.). The opticaldensity (OD) data from these cultures are shown in FIG. 6. TABLE 2 KBAPformulations used in osteoconductivity testing Vol. of 10% Vol. of 5%Vol. of 2% Vol. of 0.5 mg/mL Vol. of Cefazolin α + γ-keratose chitosanlactate sodium alginate HA + TCP transglutaminase 0.15% CaCl₂ No. (mg)gel used (μl) used (μL) used (μL) (mg) added (μL) used (μL) 4 2 500 5050 40 + 10 100 50 5 1 500 50 50 40 + 10 100 50 6 0 500 50 50 40 + 10 10050 7 0 500 50 50 40 + 10 100 50 8 0 500 50 50 40 + 10 100 50 9 0 500 5050 40 + 10 100 50

These data indicate that the KBAP formulations are capable of acting asosteoconductors as they will support the growth of osteoblasts inculture.

The ability of KBAP to recniit osteogenic cells through a chemotacticmechanism (i.e. osteoconduction) was assessed using a porous cellmembrane insert. This technique determines the extent of cell migrationthrough the porous membrane in response to a concentration gradient ofchemotactic agent(s). In these experiments, KBAP was placed in serumfree media at 37° C. and the supernatant removed at different timepoints such as 1, 3, 6, and 24 hours after immersion. This KBAP“extract” was placed in culture wells and the inserts place on top (notshown). Bovine osteoblasts were seeded at approximately 2×10⁴ cells/wellinto the upper chamber and incubated at 37° C., 5% CO₂, and 95% relativehumidity.

Cells cultured in the presence of serum free media alone was used as acontrol. After 6 and 20 hours of incubation, the membrane was fixed withglutaraldehyde and dehydrated with an alcohol gradient. The morphologiccharacteristics of the cells was examined by an environmental scanningelectron microscopy (SEM; model N-2600 Hitachi, Japan).

The effect of the KBAP extract on osteoblast migration is apparent inthe micrographs shown in FIG. 7. These data suggest that the KBAPpossesses soluble molecules that are capable of affecting osteoblastmigration. Media with no KBAP extract showed few cells on the top(“face”) surface of the membrane and none on the bottom (“back”). Mediathat had been used to extract KBAP for 6 and 24 hours showed more cellson the top surface, with 24 hours of extracting appearing to be slightlymore effective. More importantly, cells migrated through the pores tothe bottom surface of the membrane in the presence of KBAP extract after20 hours of culture. These data indicate the chemotactic potential ofKBAP's soluble fraction and the latent osteoinductivity of keratinbiomaterials.

4. Conclusions.

We evaluated three forms of keratin biomaterials for suitability in abone graft substitute formulation termed keratin bioceramic antibioticputty or KBAP. A low hydrolysis method for the extraction of keratinsfrom human hair fibers was developed such that the protein possessed thedesired viscoelastic characteristics. We discovered that when highmolecular weight keratins were obtained, they were capable ofself-assembling into fibrous micro-architectures that are conducive tocell infiltration and growth.

We further improved the physical characteristics of this self-assembledhydrogel by crosslinking strategies. From this keratin-based hydrogel,we developed a malleable KBAP prototype formulation that contained adrug delivery system capable of releasing antibiotics.

The KBAP prototype formulation was tested for antibiotic release in anin vitro model using S. aureus and was shown to effectively kill thisspecies of bacteria. We further characterized the antibiotic release bydetermining the in vitro release kinetics. The biocompatibility ofseveral different KBAP formulations was demonstrated in vitro usingbovine osteoblasts, and the osteoinductivity of human hair keratins wasshown using a cell migration assay. These data show the feasibility offormulating a malleable bone graft substitute with antibiotic releasefrom a keratin biomaterial.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof; The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A malleable bone graft composition, comprising: (a) from 1 to 90percent by weight keratose; (b) from 1 to 90 percent by weightparticulate filler; (c) from 0.001 to 5 percent by weight antibiotic;and (d) water to balance; said composition having a viscosity of atleast 3 centipoise at a temperature of 37° C.
 2. The composition ofclaim 1, wherein said keratose is alpha keratose, gamma keratose, ormixtures thereof.
 3. The composition of claim 1, wherein said keratoseis a mixture of alpha keratose and gamma keratose.
 4. The composition ofclaim 1, wherein said keratose comprises from 10 to 90 percent by weightalpha keratose and from 90 to 10 percent by weight gamnma keratose. 5.The composition of claim 1, wherein said keratose is crosslinkedkeratose.
 6. The composition of claim 1, wherein said crosslinkedkeratose is produced by the process of combining said keratose withtransglutaminase in the presence of a calcium initiator.
 7. Thecomposition of claim 1, further comprising from 0.001 to 5 percent byweight bone morphogenic protein.
 8. The composition of claim 1, whereinsaid particulate filler is osteoconductive.
 9. The composition of claim1, wherein said particulate filler is selected from the group consistingof tetracalcium phosphate, tnrcalcium phosphate, calcium alkaliphosphate ceramic, bioglass, calcium carbonate, calcium hydroxide,calcium oxide, calcium fluoride, calcium sulfate, magnesium hydroxide,hydroxyapatite, calcium phosphorus apatite, magnesium oxide, magnesiumcarbonate, magnesium fluoride, collagen, other resorbable biocompatiblematerials and mixtures thereof.
 10. The composition of claim 1, whereinsaid particulate biocompatible filler comprises hydroxyapatite,tricalcium phosphate, or a mixture thereof.
 11. The composition of claim1, wherein said antibiotic is selected from the group consisting ofcefazolin, vancomycin, gentamycin, erythromycin, bacitracin, neomycin,penicillin, polymycin B, tetracycline, biomycin, chloromycetin,streptomycin, ampicillin, azactam, tobramycin, clindamycin, gentamicinand combinations thereof.
 12. The composition of claim 1, wherein saidcomposition is sterile.
 13. The composition of claim 12 packaged in asterile container.
 14. A lyophilized or freeze-dried composition whichupon reconstitution with water or saline solution produces a compositionof claim
 1. 15. A method of treating a fracture in a subject in needthereof, comprising contacting a composition of claim 1 to said fracturein a treatment-effective amount.